Method for recovering proteins from the interstitial fluid of plant tissues

ABSTRACT

A method for extracting proteins from the intercellular space of plants is provided. The method is applicable to the large scale isolation of many active proteins of interest synthesized by plant cells. The method may be used commercially to recover recombinantly produced proteins from plant hosts thereby making the large scale use of plants as sources for recombinant protein production feasible.

This application is a continuation of U.S. patent application Ser. No.10/632,240 filed Aug. 1, 2003, now U.S. Pat. No. 6,841,659; which is acontinuation of U.S. patent application Ser. No. 10/119,330 filed Apr.8, 2002, now U.S. Pat. No. 6,617,435; which is a continuation ofapplication Ser. No. 09/726,648, filed Nov. 28, 2000, now U.S. Pat. No.6,441,147; which is a continuation of application Ser. No. 09/500,554,filed Feb. 9, 2000, now U.S. Pat. No. 6,284,875; which is a continuationof U.S. application Ser. No. 09/132,989, filed Aug. 11, 1998, nowabandoned.

FIELD OF THE INVENTION

The present invention relates generally to the field of proteinproduction and purification. More specifically, the present inventionrelates to a method for isolating commercial scale quantities of highlyconcentrated, active proteins from the intercellular material of plantsvia a vacuum and centrifugation process which does not destroy the plantmaterial, permitting secondary protein extraction from the plantmaterial.

BACKGROUND OF THE INVENTION

There are many examples of valuable proteins that are useful inpharmaceutical and industrial applications. Often these molecules arerequired in large quantities and in partially or highly purifiedformulations to maintain product quality and performance. Plants are aninexpensive source of proteins, including recombinant proteins. Manyhave proposed the desirability of producing proteins in large amounts inplants. However, the problems associated with extracting and processingproducts from homogenized plant tissues as well as purifying andrecovering the recombinant protein product have been recognized assubstantial. Austin et al. Annals New York Academy of Science,721:234–244 (1994). These problems represent major impediments tosuccessful recombinant protein production in plants on a large andcommercially valuable scale.

Plant cells are thought to synthesize proteins on the membranes of theendoplasmic reticulum and transport the proteins synthesized to the cellsurface in secretory vesicles formed at the Golgi apparatus. Adiscussion of the topic is provided by Jones et al., New Phytology,111:567–597 (1989). Significant research has been devoted to elucidatingthe specific mechanisms related to protein secretion for severalparticular proteins in specific plant tissues or cell cultures. Examplesof such efforts are presented by Herbers et al., Biotechnology 13:63–66(1995), Denecke et al., The Plant Cell 2:51–59 (1990), Melchers et al.,Plant Molecular Biology 21:583–593 (1993) and Sato et al., Biochemicaland Biophysical Research Communications 211(3):909–913 (1995). In thecase of proteins not secreted into the plant cell apoplasm orintercellular space, a mechanism for lysing the plant cell wall must beutilized in order to release and capture the protein of interest. Plantcells must be exposed to very high shear forces in order to break thecell walls and lyse cellular membranes to release intracellularcontents. Proteins of interest, whether recombinantly produced ornaturally produced by the subject plant, are thereby exposed to ahostile chemical environment and are particularly subject to oxidativeand proteolytic damage due to the exposure of the product to enzymes andsmall molecules that were compartmentalized before homogenization of thetissue. In addition, most of the other total cellular protein is mixedwith the protein of interest creating formidable purification problemsif such a cell lysis procedure is performed. In order to use thebiosynthetic capacity of plants for reliable protein production, aprocess to obtain specific proteins that can be secreted into theintercellular space (apoplasm) of plant tissues is desirable. Such aprocedure would forego the need for homogenization. If such a procedureis performed, the fraction of plant material containing one or moreproteins of interest might be obtained without homogenization.Therefore, such a procedure provides that the plant extract is enrichedfor the particular protein of interest, and the protein is protectedfrom some chemical and enzymatic degradation.

Since the valuable proteins and products of interest are partitioned orsecreted into the interstitial spaces, vacuum pressure facilitates theintroduction of infiltration medium into the interstitial space.Similarly, various forces can be applied to remove the retained fluid.Centrifugal force of 1,000×G is effective. Using gravity, the retainedfluid can be collected in a trap under vacuum. With or without vacuuminfiltration of a buffer, the enzyme can be recovered by freezing thetissue, thawing and applying a physical press to recover the fluid.However, such a procedure results in an undesirable increased cellularlysis.

Genetically modified plants are a reliable source for the production ofrecombinant proteins. Because the biological product is accumulatedunder nonsterile growth conditions and the production may be scaled tothe quantities desired in a relatively inexpensive manner, it isfeasible to exploit a dilute but enriched source such as theinterstitial fluid fraction as a S source for harvesting proteins ofinterest on an industrial scale. A variety of proteins of interest maybe harvested from recombinant plant sources, however, highly active,pharmaceutical quality enzymes, cytokines and antibodies areparticularly valuable products that can be developed by this process.

SUMMARY OF THE INVENTION

The present invention features a method for extracting highlyconcentrated, active proteins from the intercellular space of plants.The intercellular space consists of a matrix of fluid, protein and cellwall carbohydrates. The method is applicable to the large,commercial-scale isolation of proteins desired from plant cells whethersuch proteins are naturally occurring or are produced by recombinanttechnology. The vacuum and centrifugation process, as explained below,allows extraction of protein from the interstitial fluid of the plantwithout destroying the plant material, permitting further extraction ofdesired protein from the plant material.

In a broad aspect, the method comprises infiltrating plant leaves with abuffer solution by subjecting submerged plant foliage to a substantiallyvacuum environment, removing the excess liquid from the plant foliageafter exposing the foliage to the substantially vacuum environment, andcentrifuging the foliage to obtain the interstitial fluid. As a resultof such a procedure, large amounts of desirable proteins may be removedfrom the intercellular space of plants thereby making it feasible toisolate naturally-occurring proteins from plant foliage and making itpossible to produce recombinantly the desired proteins in plants andrecover the same in commercially valuable quantities withouthomogenizing the plant foliage or otherwise significantly lysing theplant cells themselves. This material is referred to as an interstitialfluid, hereinafter “IF”, IF extract.

In one embodiment, the subject plant leaves are disected completely orsubstantially down the midrib (substantially in halves) before exposingthem to the buffer solution. In another preferred embodiment, the leavesand buffer solution are subjected to a vacuum pressure of about 200 upto 760 mm Hg. Even more preferably, the leaves and buffer solution aresubjected to a vacuum pressure of about 400 up to 760 mm Hg. And mostoptimally, the leaves and buffer solution are subjected to a vacuumpressure of up to about 760 mm Hg. In yet other preferred embodiments,the leaves are subjected to a low speed centrifugation having a G-forcerange of about 50 to 5,000×G or less after the excess buffer solution isremoved. Most preferably, the leaves are subjected to centrifugationhaving a G-force of about 2,000×G.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 General Overview Of The IF Extraction Process

FIG. 2 Batch Vessel Infiltration

FIG. 3 Continuous Vacuum Infiltration

FIG. 4 Plasmid map of TT01A 103L (SEQ ID NO: 1)

FIG. 5 Viral cDNA Sequence of Plasmid TT01A 103L

DETAILED DESCRIPTION OF THE INVENTION

The present invention features a method for extracting proteins from theintercellular space of plants. The method is applicable to thelarge-scale commercial isolation of highly concentrated and activeproteins desired from plant cells whether such proteins are naturallyoccurring or are produced by recombinant technology, including the useof plant viral vectors or the use of transgenic plants. The vacuum andcentrifugation process of the present invention permits extraction ofprotein from the intercellular space without destroying the plantmaterial, thereby permitting further secondary extraction of desiredproteins from the plant material. These proteins derived from thesecondary extraction process can be either the same or different asthose proteins purified from the IF fluid.

The method generally comprises the steps of infiltrating plant foliagewith a buffer solution by subjecting the submerged plant foliage to asubstantially vacuum environment, removing the excess liquid from theplant foliage after exposing the foliage to the substantially vacuumenvironment, and centrifuging the foliage. As a result of suchprocedure, large amounts of desirable proteins may be removed from theintercellular space of plants thereby making it feasible to isolate bothnaturally-occurring and recombinantly produced proteins from plantfoliage in commercial-scale quantities without homogenizing the plantcells, allowing secondary extraction of desired protein from the plantcell material.

Work has been conducted in the area of developing suitable vectors forexpressing foreign DNA in plant hosts. Ahlquist, U.S. Pat. No. 4,885,248and U.S. Pat. No. 5,173,410 describes preliminary work done in devisingtransfer vectors which might be useful in transferring foreign geneticmaterial into a plant host for the purpose of expression therein.

Additional aspects of hybrid RNA viruses and RNA transformation vectorsare described by Ahlquist et al. in U.S. Pat. Nos. 5,466,788, 5,602,242,5,627,060 and 5,500,360 all of which are herein incorporated byreference. Donson et al., U.S. Pat. No. 5,316,931 and U.S. Pat. No.5,589,367, herein incorporated by reference, demonstrate for the firsttime plant viral vectors suitable for the systemic expression of foreigngenetic material in plants. Donson et al. describe plant viral vectorshaving heterologous subgenomic promoters for the stable systemicexpression of foreign genes. Hence, the use of plants to producerecombinant proteins on a commercial scale is now possible. The presentapplication solves the problem of extracting these proteins of interestfrom the interstitial fluid of plant foliage.

Protein secretion in plants is a fundamental yet not fully understoodprocess. It is known that secreted proteins are synthesized on themembranes of the rough endoplasmic reticulum and transported to the cellsurface by secretory vesicles formed on the Golgi apparatus. Moreover,it is known that a signal peptide is required for translocation of thesecreted proteins across the endoplasmic reticulum. Proteins which aretransported into the lumen of the endoplasmic reticulum may then besecreted into the interstitial space provided they are not sorted by thecell to another compartment such as the vacuole. As knowledge about thisprocess increases, it may be possible to design recombinant proteinswhich are specifically intended for secretion into the interstitialspace of plant cells in which they are produced.

If a significant percentage (approximately 10% or greater) of the totalproduct is secreted then it may be preferable to isolate proteins ofinterest from the intercellular space of plants. Otherwise, a mechanismfor lysing the plant cell wall must be utilized in order to release andcapture the protein of interest. Plant cells must be exposed to veryhigh shear forces in order to break the cell walls and lyse cellularmembranes to release intracellular contents. Proteins of interest,whether recombinantly produced or naturally produced by the subjectplant, are thereby exposed to a hostile chemical environment and areparticularly subject to oxidative and proteolytic damage that is oftenenzymatically catalyzed. In addition, most of the other total cellularprotein is mixed with the protein of interest creating formidablepurification problems if such a cell lysis procedure is performed.

Intercellular fluid extracts have previously been prepared from vacuuminfiltrated foliage for a variety of experimental purposes. Theseextracts are comprised of proteins, both native and nonnative, as wellas other molecules. In Klement, Z. (1965) PhytopathologicalNotes:1033–1034, the growth promoting properties of the extract weredocumented using a plant pathogenic bacterial species. Using markerenzymes for the IF and cytosolic compartments of the plant leaf cell,Rathmell and Sequera (1974), Plant Physiol. 53:317–318 confirmed theenrichment of a specifically secreted protein fraction and noted theutility of these extracts in basic research studies pertaining tobiochemical and physiological investigations. Parent and Asselin (1984)Can. J. Bot. 62:564–569, characterized a number of proteins that wereinduced by pathogen stress and secreted in the IF (pathogenesis-relatedor PR proteins) and the method was applied to localize enzymaticactivities and proteins to subcellular compartments. Van den Blucke etal. (1989) PNAS 86:2673–2677; Heitz et al. (1991) Plant Physiol.97:651–656. Regalado and Ricardo (1996) Plant Physiol. 110:227–232 notedthat specific IF proteins appear to be constitutively expressed.

Depending on the buffer composition and treatment, there may be variousadditional components in IF extracts including, for example, componentsoriginating from the rough and smooth endoplasmic reticulum, the golgiapparatus, the nucleus, the vacuole, the plasma transmembrane, thecytosol, the mitochondria, the chloroplasts, peroxisomes, any associatedmembranes and organelles.

In genetically modified plants, IF extraction methods as well as othermethods have been used to demonstrate the subcellular localization of aportion of the recombinant product. Sijomns et al. (1990) Bio/Technology8:217–221; Firek et al. (1993) Plant Molecular Biology 23:861–870; Vosset al. (1995) Molecular Breeding 1:39–50; De Wilde et al. (1996) PlantScience 114:233–241. IF extracts have been used as a starting materialto purify small quantities of plant or plant pathogen-derived proteinsfor biochemical characterization. Melchers et al. (1993) Plant MolecularBiology 21:583–593; Sato et al. (1995) BBRC 211:909–913; Kinai et al.(1995) Plant Cell 7:677–688; Liu et al. (1996) Plant Science121:123–131; Maggio et al. (1996) Plant Molecular Biology Reporter14:249–259.

Therefore, there is a need to isolate an extracted material having ahigher specific activity of the active material (U activity/mg protein)and, therefore, this provides an enrichment process of IF components atcommercial scale.

It is not appreciated in the prior art that IF extracts might begenerally useful as starting material for the large scale purificationof highly active and potent biochemicals that may, for example, haveapplications as a source of human therapeutics. Often other methods ofpurification are pursued even when the product is shown to be secreted(Herbers et al. 1995, supra). The failure to develop the IF method as acommercially feasible source of recombinant protein products is due to acombination of the following factors: 1) an incomplete characterizationof the extracts, i.e. a determination of what percent of the totalrecombinant protein can be obtained by IF methods at what level ofenrichment, 2) failure by others to demonstrate suitable activity of aproduct in a highly purified form and 3) a lack of description ofindustrial-scale equipment to process reasonable quantities of biomassfor this purpose.

The present invention involves a vacuum and centrifugation process toprovide for commercial-scale protein extraction from plants. As a resultof the present invention, large amounts of active proteins of interestmay be removed from the intercellular space of plants and concentratedfor further purification thereby making it feasible to isolatenaturally-occurring and recombinantly-produced proteins from plantfoliage in commercially valuable quantities. This process has anadditional advantage in that the resulting plant tissue following IFextraction is not destroyed and may be used for recovery of othervaluable components by other means.

The foliage may be harvested in any manner that is convenient. In apreferred embodiment, the subject plant leaves are removed from theplant and are dissected completely or substantially lengthwise parallelto the midvein substantially in halves before exposing them to a buffersolution such that the ends of numerous large lateral veins are exposed.

Once the leaves are cut, they may be exposed to a buffer solution. Aroutine EDTA or Tris buffer solution is suitable, though those skilledin the art will appreciate that any buffer may be more or lessappropriate for a given plant or protein of interest. In some instances,water may be acceptable or even preferred as a solution. It is notcontemplated that the nature of the buffer solution, specific pH ortemperature are crucial to the embodiments within the scope of theinvention. However, it is generally recommended to maintain conditionswhich avoid oxidation, precipitation, proteolysis or denaturation of theone or more proteins of interest. Thus, pH, temperature, and other suchvariables should be monitored and altered as needed.

Once the leaves of the plant have been placed in a buffer solution, theyare subjected to a substantially vacuum environment. It is believed thatvacuum pressure expedites soaking of the buffer solution by the leaf. Insome embodiments, the vacuum pressure may be about 200 to 760 mm Hg.Most preferably, the leaves and buffer solution are subjected to avacuum pressure of about 400 to 760 mm Hg. The amount of vacuum pressuremay be varied within the scope of the invention. Also, the duration maybe varied within the scope of the invention, however, exposure to avacuum environment for durations of around a few seconds to 10 minuteshas proven especially effective. In some embodiments of the invention,the leaves in buffer solution are exposed to a vacuum environmentrepeatedly. It is believed that one to three separate exposures may beespecially effective. However, the number of exposures, duration ofexposure and amount of force of the vacuum may be adjusted according tothe preferences of the practitioner and to capture the most efficientembodiments of the method as it applies to specific plants and proteinsof interest. Additionally, one skilled in the art can invision thatmolecules or products of interest other than peptides and proteins couldbe recovered from the interstitial fluid using methods generallydescribed in the instant invention. For example, the methods describedin the instant invention can be used to recover lipids, carbohydrates,lipoproteins, sugars, polysaccharides, fatty acids, nucleic acids andpolynucleotides.

The plant tissue is then removed from the buffering solution. They mayor may not be subjected to a desiccation step to remove the buffer asthe need or desire dictates. The leaves may then be placed in anyconvenient geometric array for centrifugation. In preferred embodimentsthe leaves are transferred from the centrifuge by means of adiscontinuous discharge basket centrifuge rotor. When a discontinuousdischarge basket centrifuge rotor is used, an initial spin is performedto move the biomass to the wall of the rotor and then the full-speedspin is performed. In especially preferred embodiments, it iscontemplated that a large volume of leaves will be simultaneouslysubjected to the vacuum and centrifuging devices. Thus, it isanticipated that large, commercially available vacuum pumps and basketcentrifuges such as those made by Heine®, Ketna® or Sandborn® will beused in the subject method. It is especially preferred to assemble theleaves in bags for a basket centrifuge.

The leaves may then be subjected to centrifugation after the excessbuffer solution is substantially removed. In preferred embodiments, itis contemplated that low speed centrifugation is appropriate. By lowspeed centrifugation is meant about 5,000×G or less. By thecentrifugation procedure, the interstitial fluid is removed from theplant. The interstitial fluid may be collected in any convenientcollecting device, e.g., a tank, or directed to additional purificationequipment, e.g., chromatography and ultrafiltration.

Once the interstitial fluid is collected from plant leaves, the one ormore proteins of interest may be concentrated and purified according toany suitable purification procedures.

Such procedures may include but are not limited to proteinprecipitation, expanded bed chromatography, ultrafiltration, anionexchange chromatography, cation exchange chromatography,hydrophobic-interaction chromatography, HPLC, FPLC and affinitychromatography. A general discussion of some protein purificationtechniques is provided by Jervis et al., Journal of Biotechnology11:161–198 (1989), the teachings of which are herein incorporated byreference.

It is contemplated that the method of the present invention is usefulwith any and all plant tissues (such as leaves, roots, shoots, stems,flowers, fruits, embryos, seedlings) that may be treated as saturatedsolids after vacuum infiltration. For example, this may includegerminating embryos and seedlings. However, plants possessingsubstantially symmetrical leaves with a midrib may be especially usefulin the present method because the interstitial fluid may be more easilyobtained from such leaves as a result of the highly suitable morphology.In especially preferred embodiments, the plant used is tobacco sincetobacco has proven to be especially useful in producing recombinantproteins of interest on a large scale. However, it is not intended thatthe present invention be limited to any particular plant species ortissues.

The following definitions are provided merely to clarify the presentinvention:

By “vacuum environment” is meant any environment regardless of theconfines defining the same and regardless to the mechanism producing thesame in which the atmospheric pressure has been substantially reducedfrom that observed under normal conditions at sea level.

By “protein of interest” is meant any complete protein or peptide orfragment thereof whether naturally occurring in a cell or producedtherein by recombinant methods. The term is intended to encompass aminoacid sequences which are glycosylated as well as those which are notglycosylated. The term is also intended to encompass sequences which arenaturally occurring or wild type and those which have been modified ormutated, including modification to include a signaling peptide sequencewhich causes the protein to be directed to a specific compartment withinthe cell. The term is also intended to encompass protein fusions.

By “interstitial fluid” is meant the extract obtained from all of thearea of a plant not encompassed by the plasmalemma i.e., the cellsurface membrane. The term is meant to include all of the fluid,materials, area or space of a plant which is not intracellular (whereinintracellular is defined to be synonymous with innercellular) includingmolecules that may be released from the plasmalemma by this treatmentwithout significant cell lysis. Synonyms for this term might be exoplasmor apoplasm or intercellular fluid or extracellular fluid. Interstitialfluid is abbreviated in the instant invention as IF.

EXAMPLES

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limited.

These experiments demonstrate that a significant portion of the totalprotein in the leaf can be simply recovered from the interstitialfraction while enriching for purity. These experiments furtherdemonstrate that the methods are useful for the isolation of highlyactive products on a very large scale. Those skilled in the art mayoptimize the process for numerous variables specific for each proteinsuch as buffer composition and temperature, etc.

Example 1 Extraction of α-Trichosanthin Protein

α-Trichosanthin (α-TCS) is a eukaryote ribosome-inactivating enzyme thatcleaves an N-glycosidic bond in 28S rRNA. α-TCS, as well as otherribosome-inactivating proteins and conjugates are being evaluated astherapeutics for cell-directed death. In previous work we demonstratedthat plants transfected with a proprietary RNA viral vector producerecombinant α-TCS to 2% of the total soluble leaf protein with highfidelity (Kumagai et al. PNAS 90:427–430 (1993)).

Leaves from plants transfected with the vector TB2 (ATCC Deposit No.75280) were removed at the petiole and slit down the midrib into twoequal halves. To obtain a total cellular homogenate, one group ofhalf-leaves was ground in the presence of 4 volumes of detergentextraction buffer (100 mM potassium phosphate pH 6.5 mM EDTA, 10 mM,α-mercaptoethanol and 0.5% w/v sodium taurocholate) with a mortar andpestle after freezing the tissue in liquid nitrogen. To recover theinterstitial fluid (IF), the same enzyme extraction buffer wasinfiltrated into the opposing group of half-leaves by submerging thetissue and pumping a moderate vacuum (500 mm Hg). After draining offexcess buffer, the undisrupted half-leaves were rolled gently inparafilm, placed in disposable tubes and the interstitial fluid (IF) wascollected by low-speed centrifugation (1,000×G) for a period of 5–15minutes. The weight of buffer recovered from the infiltrated leaf tissueis recorded and varies from approximately one-half to equal the originalweight of the leaf. α-TCS expression in IF extracts was confirmed byWestern analysis and levels were quantified using a densitometer tracingof a Coomassie-stained gel. Total protein was determined by the methoddescribed by Bradford. Bradford, Anal. Biochem 72:248 1976.

The following data presented as Table 1 demonstrate that recombinantα-TCS, shown in previous work to retain full enzymatic activity, may besuccessfully extracted from the interstitial fluid of plant leaves usingthe present method. The IF method results in a recovery of 9% of thetotal α-TCS of the leaf at a 6-fold enrichment relative to an extractobtained by homogenization (H). The α-TCS production results may beimproved by optimizing the time post-inoculation with the viral vectorand minimizing the contamination of viral coat protein in theinterstitial fraction.

TABLE 1 Fresh Total Protein Total Protein Rprotein Total 1Rprotein %Recovery Weight Volume Conc. Protein Yield Conc. RProtein Yield RProteinX-Fold Sample (gr) (ml) (mg/ml) (mg) (mg/gr) (mg/ml) (mg) (mg/gr) In IFPurification TB2/IF 8.00 7.8 0.13 1.03 0.13 ND ND ND ND ND TB2/TCS/IF8.00 8.3 0.14 1.20 0.15 0.017 0.143 0.018 9 6 *TB2/TCS/H ND ND ND 80.00ND ND 1.600 ND ND ND *Calculated from PNAS 90: 427–430 (1993) IF =interstitial fluid extraction H = homogenization extraction ND = Notdetermined

Example 2 Extraction of Amylase Protein

Amylase (AMY) is an important industrial enzyme used to degrade starch.Leaves from plants transfected with the vector TT01A 103L were removedat the petiole and slit down the midrib into two equal halves. Theplasmid map of TT01A 103L is shown in FIG. 4. The viral cDNA sequence ofplasmid TT01A 103L is shown in FIG. 5. To obtain a total cellularhomogenate, one group of half-leaves was ground in the presence of 4volumes of detergent extraction buffer (100 mM potassium phosphatepH6.5, 5 mM EDTA, 10 mM, α-mercaptoethanol and 0.5% w/v sodiumtaurocholate) with a mortar and pestle after freezing the tissue inliquid nitrogen. To recover the interstitial fluid (IF), the same enzymeextraction buffer was infiltrated into the opposing group of half-leavesby submerging the tissue and pumping a moderate vacuum (500 mm Hg).After draining off excess buffer, the undisrupted half-leaves wererolled gently in parafilm, placed in disposable tubes and theinterstitial fluid (IF) was collected by low-speed centrifugation(1,000×G for about 15 minutes). The weight of buffer recovered from theinfiltrated leaf tissue is recorded and varies from approximatelyone-half to equal the original weight of the leaf. AMY expression in IFextracts was quantified using a commercially available enzyme assayreagents and protocol. Total protein was determined by the methoddescribed in Bradford, Anal. Biochem 72:248 1976. The AMY enzyme assayis described in Sigma Procedure No. 577.

The following data presented as Table 2 demonstrate that activerecombinant AMY may be successfully extracted from the interstitialfluid of plant leaves using the present method. The IF method results ina recovery of 34% of the total AMY activity of the leaf at a 27-foldenrichment relative to an extract obtained by homogenization (H). TheAMY production results may be improved by optimizing the timepost-inoculation with the viral vector and minimizing the contaminatingviral coat protein from the intercellular fraction.

Example 3 Extraction of Glucocerebrosidase Protein

Glucocerebrosidase (GCB), either derived from human placental tissue ora recombinant form from Chinese hamster ovary cells (CHO), is presentlyused in an effective but costly treatment of the heritable metabolicstorage disorder known as Gaucher disease. We combined a dual promoterfrom Cauliflower Mosaic Virus (35S), a translational enhancer fromTobacco Etch Virus and a polyadenylation region from the nopalinesynthetase gene of Agrobacterium tumaciens with the native human GCBcDNA to create plasmid pBSG638. These expression elements are widelyused to provide the highest possible constitutive expression ofnuclear-encoded genes in plants.

Using a standard Agrobacterium-mediated transformation method, weregenerated 93 independent kanamycin-resistant transformants from leafdiscs of four different tobacco cultivars (the TO generation). InWestern blots of total protein extracts, cross-reacting antigen wasdetected in 46 of these TO individuals with antibody raised againsthuman glucocerebrosidase. Specificity of the plant-expressed recombinantenzyme was confirmed by hydrolysis of 14C-radiolabeled glucosylceramide.According to these expression results the rGCB positive transformantswere ranked into moderate (A), low (B) and negligible (C) activitygroups.

We also found reaction conditions to preferentially inhibit rGCB enzymeactivity in the presence of plant glucosidases using the suicidesubstrate conduritol B-epoxide (CBE). Total glucosidase activity, andrGCB activity were measured by hydrolysis of the fluorescent substrate4-methylumbelliferyl glucopyranoside (4-MUG) with and without CBE.Leaves from transgenic plants were removed at the petiole and disecteddown the midrib into two equal halves to make a convenient size leafmaterial for the equipment used. To obtain a total cellular homogenate,one group of half-leaves was ground in the presence of 4 volumes ofdetergent extraction buffer (100 mM potassium phosphate pH 6.5 mM EDTA,10 mM, α-mercaptoethanol and 0.5% w/v sodium taurocholate) with a mortarand pestle after freezing the tissue in liquid nitrogen. One of ordinaryskill in the art could readily envision a buffer wherein the EDTA issubstituted with other chelaters such as EGTA and citrate. One ofordinary skill in the art could readily envision a buffer solutionwherein α-mercapto ethanol

TABLE 2 Fresh Total Protein Total Protein Rprotein Total 2Specific3RProtein % Recovery Weight Volume Conc. Protein Yield Conc. RProteinYield Yield RProtein X-Fold Sample (gr) (ml) (mg/ml) (mg) (mg/gr) (Uml)(U) (U/gr) (U/gr) In IF Purification Amylase/IF 1.76 1.8 0.22 0.39 0.220.319 0.57 0.33 1.463 34 27 Amylase/H 1.76 5.8 5.40 31.33 17.80 0.2901.68 0.96 0.054 ND 1 IF = Interstitial fluid extraction H =homogenization extraction ND = Not determinedis substituted by other antioxidants including ascorbate, sodiummetabisulfite and dithiothreitol. One of ordinary skill in the art canreadily invision that a buffer solution could substitute the sodiumtaurocholate with other detergents including: SDS, Triton®(t-octylphenoxypolyethoxyethanol), Tween® (fatty acid esters ofpolyoxyethylene sorbitan), phospholipids, bile salts, sodiumdeoxycholate and sodium lauryl sulfate. To recover the interstitialfluid (IF), the same enzyme extraction buffer was infiltrated into theopposing of group of half-leaves by submerging the tissue and pumping amoderate vacuum (500 mm Hg). After draining off excess buffer, theundisrupted half-leaves were rolled gently in parafilm, placed indisposable tubes and the interstitial fluid (IF) was collected bylow-speed centrifugation (1,000×G) for about 15 minutes. The weight ofbuffer recovered from the infiltrated leaf tissue is recorded and variesfrom approximately one-half to equal the original weight of the leaf.Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 Mpotassium phosphate, 0.15% Triton-X100, 0.125% sodium taurocholate, 0.1%bovine serum albumin, pH 5.9 with and without CBE. Total glucosidaseactivity and rGCB activity were measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl glucopyranoside. One unit ofactivity is defined as the amount of enzyme required to catalyze thehydrolysis of 1 nmol of substrate per hour. Total protein was determinedusing the Bio-Rad Protein Assay® based on the method of Bradford(Bradford, M., Anal. Biochem. 72:248; 1976)

The following data presented as Table 3 demonstrate that activerecombinant GCB may be successfully extracted from the interstitialfluid of plant leaves using the present method. The IF method results ina recovery of 22% of the total GCB activity of the leaf at a 18-foldenrichment relative to an extract obtained by homogenization.

Example 4 Extraction of Avian Interferon Type II (Gamma)

Avian (chicken) interferon type II (gamma) has been expressed and activeenzyme extracted from the interstitial space of Nicotiana benthamianaand Nicotiana tabacum. The interferon could be efficiently extractedfrom plants grown in the field or greenhouse using either gram(bench-scale extraction) or Kg (pilot-scale extraction) quantities ofplant tissue.

Actively growing N. benthamiana or N. tabacum were inoculated witheither infectious transcripts or virion of a recombinant plant constructas described by Donson et al., supra, harboring the chicken interferongamma gene. Avian interferon was extracted from systemically infectedleaves 10 days to 3 weeks post inoculation.

Example 4a

For bench-scale extractions, systemically infected leaves (3–80 grams)were detached from the plant at the leaf base, weighed, and placed in anappropriate sized beaker. The leaf material was completely covered witha buffered solution (100 mM Tris-HCl pH 7.5 buffer containing 5 mM MgCl₂and 2 mM EDTA). The immersed leaves were covered with a Nalgene vacuumjar and a vacuum was pumped (720 mm Hg) and held for 2 minutes and thenrapidly released. This vacuum infiltration was then repeated for a totalof two cycles. Following the vacuum infiltrations, the leaves wereremoved from the beaker and surface buffer was removed from the leaves'surface by blotting between absorbent paper. Leaves were placed in a 250ml bottle, containing a supported mesh which allows for the separationand recovery of the IF from the leaf material. The interstitial fluid(IF) was recovered from the vacuum infiltrated leaves by centrifugation(3,000×G, 15 minutes).

Example 4b

For pilot-scale extractions, systemically infected leaves from fieldgrown plants were stripped off the stalks by hand and weighed. Five kgof leaves were placed into polyester mesh bags (Filtra-Spec®,12-2-1053), and two×5 kg bags of leaves were placed into a metal basketThe metal basket containing the leaf material was placed in a 200 LMueller® vacuum tank containing ˜50 liters of buffered solution (100 mMTris-HCl pH 7.5 buffer containing 5 mM MgCl₂ and 2 mM EDTA). A 70 lb.stainless steel plate was placed over the leaves/bags to assure completeimmersion. A vacuum was pumped to 27 inches Hg and held for 1 minute andthen rapidly released. This vacuum infiltration was then repeated for atotal of two cycles. Following the vacuum infiltrations, the leaves andbasket were removed from the vacuum tank. The bags containing the vacuuminfiltrated leaves were allowed to gravity drain surface buffer for ˜10minutes, prior to centifugation. The interstitial fluid (IF) wasrecovered from the vacuum infiltrated leaves by centrifugation (1,800×G,30 minutes) using a Heine® basket centrifuge (bowl dimensions, 28.0 inchdiameter×16.5 inches). Collected IF was filtered through a 25 μm,Rosedale® sock filter and then through a 5 μm, Campbell Waterfilter®cartridge filter and then stored at 4° C., prior to analysis.

The amount of interferon protein in an IF extract was determined byquantitative immunoblotting procedures using specific antisera to aviantype II interferon and E. coli produced type II interferon in knownquantities as standard. Based on quantitative immunoblotting, andpartial purification, we estimate the specific activity of interferon inN. benthamiana IF at or near 10⁷ U/mg which is essentially equal tointerferon isolated from native sources. Biological activity wasdetermined by the nitrous oxide (NO) release assay as described inLowenthal, J. W., Digby, M. R. and York, J. J. Production ofInterferon-y by Chicken T Cells, J. Interferon and Cytokine Res. (1995)15:933–938. Specificity of activity was determined by pre-incubation ofIF fluid with a neutralizing antibody followed by measuring activity inthe NO release assay.

TABLE 3 Fresh Total Protein Total Protein Rprotein Total SpecificRprotein % Recovery Weight Volume Conc. Protein Yield Conc. RproteinYield Yield Rprotein X-Fold Sample (g) (ml) (mg/ml) (mg) (mg/gr) (Uml)(U) (U/g) (U/g) In IF Purification GCB/IF 2.48 1.9 0.24 0.45 0.18 7201368 552 3007 22 18 GCB/H 2.08 8.1 3.89 31.48 15.13 653 5289 2543 168 ND1 IF = Interstitial fluid extraction H = homogenization extraction ND =Not determined

TABLE 4 Greenhouse: Av. Tissue Interferon Plant type Amt. protein yield¹Yield activity² N. benthamiana 3–60 g   1 mg/100 g fresh wt ~30,000 U/mlIF N. tabacum   20 g 0.1 mg/100 g fresh wt  ~3,000 U/ml IF cv. MD609Nitabacum   20 g 0.1 mg/100 g fresh wt  ~3,000 U/ml IF TI231

TABLE 5 Field: Greenhouse Av. Tissue Plant type Amt. Interferon proteinyield¹ Yield activity² N. tabacum cv 80 g  0.05 mg/100 g fresh wt ND*TI264 N. tabacum cv 10 kg 0.01 mg/100 g fresh wt ~200 U/ml IF** TI264¹Interferon protein yield was estimated by quantitative immunoblotting.²Interferon activity was determined by the NO release assay as describedby Lowenthal et al. supra *Not determined **Activity estimates containedsome lack of specificity (activity not neutralized by specific antibody)in NO release assay.

Example 5 Extraction of Mouse scFv Protein

Actively growing N. benthamiana were inoculated with infectioustranscripts of a recombinant plant construct as described by Donson etal., supra, harboring a scFv protein from the 38C13 mouse lymphoma.Mouse 38C13 scFv protein was extracted from systemically infected leaves11–14 days post inoculation.

Systemically infected leaves (3–80 grams) were detached from the plantat the leaf base, weighed, and placed in an appropriate sized beaker.The leaf material was completely covered with a buffered solution (100mM Tris-HCl pH 7.5 buffer containing 10 mM MgCl₂ and 2 mM EDTA). Theimmersed leaves were covered with a Nalgene vacuum jar and a vacuum waspumped to 700 mm Hg, and held for 2 minutes and then rapidly released.This vacuum infiltration was then repeated for a total of two cycles.Following the vacuum infiltrations, the leaves were removed from thebeaker and surface buffer was removed from the leaves' surface byblotting between absorbent paper. The interstitial fluid (IF) wasrecovered from the vacuum infiltrated leaves by centrifugation (3,000×G,15 minutes). Leaves were centrifuged in a 250 ml bottle, containing asupported mesh which allows for the separation and recovery of the IFfrom the leaf material. The IF fluid containing the scFv protein wasfiltered through a 0.2 μm membrane and stored at −80° C.

The product and purification of 38C13 scFv protein from plant IF fluidwas determined by Western analysis using S1C5, a monoclonalanti-idiotype antibody which recognizes native 38C13 IgM protein. TheS1C5 antibody cross reacted with a 30 KD protein of the expected size of38C13 scFv and a 60 KD protein, which is the correct size for aspontaneously assembling scFv dimer. No cross reactivity to plantproteins in IF extracts prepared from control infected plants wasobserved.

The quantity of plant-produced 38C13 scFv protein recovered from IFextracts was measured by S1C5 ELISA. Leaf IF extracts were determined tocontain 20–60 μg of 38C13 scFv protein/ml IF fluid or 11–30 μg of 38C13scFv protein/g fresh weight. Since ELISA conditions favor anti-idiotyperecognition in solution, it is concluded that the major fraction of38C13 scFv isolated from plant IF fluid is soluble and properly folded.

Example 6 Extraction of Secretory Immunoglobulin from Transgenic Tobacco

Leaves from transgenic, SIgA-G expressing N. tabacum (15 grains),(Science, 268:716, 1995), were detached from the plant at the leaf base,weighed, and placed in an appropriate sized beaker. The leaf materialwas completely covered with a buffered solution of either 100 mMTris-Hcl pH 7.5 buffer containing 10 mM MgCl₂, 2 mM EDTA and 14.3 mM2-mercaptoethanol or 100 mM potassium phosphate, pH6.0, 5 mM EDTA, 10.0mM 2-mercaptoethanol and 0.5% taurocholic acid). The immersed leaveswere covered with a Nalgene® vacuum jar and a vacuum was pumped to 750mm Hg, and held for 1 minute and then rapidly released. Following thevacuum infiltrations, the leaves were removed from the beaker andsurface buffer was removed from the leaves' surface by blotting betweenabsorbent paper. The interstitial fluid (IF) was recovered from thevacuum infiltrated leaves by centrifugation (1500×G, 15 minutes). Leaveswere centrifuged in a 250 ml bottle, containing a supported mesh whichallows for the separation and recovery of the IF from the leaf material.

Protein immunoblots of the IF extracts were prepared under reducingconditions. Ig was detected in the immunoblots using goat anti-mouse IgAconjugated to horseradish peroxidase. Approximately 10% of the IgApresent in the plant was detected in the IF extracts. There was novisible difference in the quantity of Ig in the IF fractions producedusing the different buffers described above. No cross reactivity toplant proteins in IF extracts prepared from control plants was observed.

Example 7 Pilot Scale Purification of Glucocerebrosidase from theIntercellular Fluid of Tobacco

MD609 leaf tissue (1–2 kilograms) of transgenic tobacco expressing thelysosomal enzyme glucocerebrosidase was harvested, the mid vein removedand the tissue weighed. Tissue was submerged with 24 volumes of buffer(0.1 M KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10 mM2-mercaptoethanol) using an infiltration vessel that accommodatesseveral kilograms of leaf tissue at one time. A perforated metal platewas placed on top of tissue to weigh down the tissue, and a vacuum waspumped to 620–695 mm Hg for 1–2 minutes×3. The vacuum was releasedbetween subsequent applications. Tissue was rotated and the vacuumreapplied to achieve complete infiltration. Multiple applications of thevacuum without isolating the interstitial fluid constitutes a singleinfiltration procedure. An indication of complete infiltration is adistinct darkening in color of the underside of the leaf tissue. Excessbuffer on the tissue was drained. The interstitial fluid was releasedfrom the tissue by centrifuging the tissue in a basket rotor (10in.×4.25 in., Inter Test Equipment Services, San Jose, Calif./BiosourceDesign 25-0611000) at 4200 RPM (2500×G) for 10 minutes. The interstitialfluid was collected by aspiration (IF-1). Alternatively, the leaf tissuecan be re-infiltrated by placing the leaves back in the infiltrationvessel in the same buffer used above and the process repeated (IF-2).The second infiltration does not require as many cycles of vacuuminfiltration and vacuum release. Additionally, the buffer may be drainedfrom the infiltration vessel (spent buffer) and pooled with the 1st and2nd IF fractions. Collectively, IF-1, IF-2 and spent buffer constitutesthe IF pool. The volume of interstitial fluid collected from theinfiltrated leaf tissue was between 50–100% of the leaf tissue by weightdepending on the number of infiltrations carried out.

Recombinant GCB was purified by loading the dilute IF (feed stream)directly on a Pharmacia Streamline 25® column containing PhenylStreamline® resin. Expanded bed chromatography enabled us to capture,clarify and concentrate our protein in one step without the need forcentrifugation and/or microfiltration steps. The column was equilibratedand washed until the UV-signal on the recorder returned to baseline with25 mM citrate, 20% ethylene glycol, pH 5.0; bound enzyme was eluted with25 mM citrate, 70% ethylene glycol. The eluted material was furtherpurified on a cation exchange resin, SP Big Beads® (Pharmacia),equilibrated in 25 mM citrate, 75 mM NaCl, pH 5.0. GCB was eluted witheither a step gradient of 25 mM citrate, 0.5 M NaCl, 10% ethyleneglycol, pH 5.0 or a linear gradient of 75 mM–0.4 M NaCl in 25 mMcitrate, pH 5.0. All chromatography steps were carried out at roomtemperature.

Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 MPotassium Phosphate, 0.15% Triton-X100, 0.125% sodium taurocholate, 0.1%bovine serum albumin, pH 5.9 with and without CBE. Total glucosidaseactivity and rGCB activity were measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl glucopyranoside. One unit ofactivity is defined as the amount of enzyme required to catalyze thehydrolysis of 1 nmol of substrate per hour. Total protein was determinedusing the Bio-Rad Protein Assay based on the method of Bradford(Bradford, M. Anal. Biochem. 72:248; 1976).

Typically from 1 kilogram of leaves where IF-1 alone was collected weobtained 4 million units of GCB at a specific activity of 20,000. TheUnits/kg increased to 6 million with a lower specific activity of 10,000when IF Pool was collected (IF-1, IF-2 and spent buffer).

Table 6 below contains data that is representative of severalexperiments.

Example 8 Ultrafiltration/Concentration of Intercellular Fluid fromTobacco Expressing Glucocerebrosidase

2.3 kilograms of MD609 leaf tissue from transgenic tobacco expressingthe lysosomal enzyme glucocerebrosidase was harvested, the mid veinremoved and the tissue weighed. Tissue was submerged with 2–4 volumes ofbuffer (0.1 M KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10mM 2-mercaptoethanol) in an infiltration vessel that accommodatesseveral kilograms of leaf tissue at one time. A perforated metal platewas placed on top of tissue to weigh down the tissue. A vacuum waspumped to 620–695 mm Hg for 1–2 minutes×3. The vacuum was releasedbetween subsequent applications. Tissue was rotated and the vacuumreapplied to achieve complete infiltration. Excess buffer on the tissuewas drained. The interstitial fluid was released from the tissue bycentrifuging the tissue in a basket rotor (10 in.×4.25 in., IntertestEquipment Services, San Jose, Calif./Biosource Design 25-0611000) at4200 RPM (2500×G) for 10 minutes. The interstitial fluid was collectedby aspiration (IF-1). The leaf tissue was re-infiltrated by placing theleaves back in the infiltration vessel in the same buffer used above andthe process repeated (IF-2). The buffer was drained from theinfiltration vessel (spent buffer) and pooled with the 1st and 2nd IFfractions. Collectively, IF-1, IF-2 and spent buffer constitutes the IFpool. The IF pool was filtered through Miracloth and then concentrated 6fold by passing the IF pool through a 1 sq. ft. spiral membrane (30Kmolecular weight cutoff) using an Amicon RA 2000® concentrator equippedwith an LP-1 pump.

TABLE 6 GCB-Lab Pilot Scale IF Process Fresh Total Protein Total ProteinGCB Total Weight Vol Conc. Protein GCB Yield Conc GCB Units/kgSample/Fraction # (Grams) (ml) (mg/ml) (mg) (mg) (mg/g) (U/ml) (Units)Tissue IF1 1045 930 0.236 219 2.91 0.21 4,692 4,363,544 4,175,640 PhenylStreamline 1045 400 0.065 26 2.47 0.025 9,276 3,710,467 3,550,6864/30/97 IF1,2 & Spent 1027 4020 0.29 1166 4.32 1.135 1,611 6,478,2016,307,888 buffer IF1,2 & SB Phenyl 1027 2330 0.29 676 2.5 0.658 1,6113,752,778 3,656,064 SL column load Phenyl Streamline 1027 400 0.078 312.36 0.03 8,858 3,543,390 3,450,234 eluted material SP Big Beads eluant1027 70 0.078 5 1.72 0.005 36,952 2,586,674 2,518,670 eluted material SpActivity % GCB Step Step Total Total nmol/hr % of total RecoveryPurification Recovery Purification Sample/Fraction # (U)/mg Protein =GCB (%) fold (%) fold IF1 19,881 1.33 100 1 100 1 Phenyl Streamline142,710 9.51 85 7.2 85 7.2 4/30/97 IF1,2 & Spent 10,047 0.67 100 1 100 1buffer IF1,2 & SB Phenyl 10,047 0.67 100 1 100 1 SL column load PhenylStreamline 113,570 7.57 94.4 11.3 94.4 11.3 eluted material SP Big Beadseluant 473,750 31.58 73 4.2 68.9 47.2 eluted material IF = interstitialfluid

Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 Mpotassium phosphate, 0.15% Triton-X100, 0.125% sodium taurocholate, 0.1%bovine serum albumin, pH 5.9 with and without CBE. Total glucosidaseactivity and rGCB activity were measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl glucopyranoside. One unit ofactivity is defined as the amount of enzyme required to catalyze thehydrolysis of 1 nmol of substrate per hour. Total protein was determinedusing the Bio-Rad Protein Assay® based on the method of Bradford(Bradford, M. Anal. Biochem. 72:248; 1976). See Table 7 below.

Example 9 Pilot Scale Purification of Glucocerebrosidase from theIntercellular Fluid of Field Grown Tobacco

100 kilograms of MD609 leaf tissue from transgenic tobacco expressingthe lysosomal enzyme glucocerebrosidase was harvested from the fieldeach day for a period of two weeks.

The tissue was stripped off the stalks by hand and weighed. Fivekilograms of leaves were placed into polyester bags (Filtra-Spec®,12-2-1053) and four×5 kg bags of leaves were placed into a metal basket.The metal basket containing the leaf material was placed in a 200 literMueller vacuum tank containing ˜100 liters of buffered solution (0.1KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10 mM2-mercaptoethanol). A 70 lb. stainless steel plate was placed over theleaves/bags to assure complete immersion. A vacuum was pumped to 695 mmHg, held for 1 minute and then rapidly released. This vacuuminfiltration was repeated for a total of two cycles. Multipleapplications of the vacuum without isolating the interstitial fluidconstitutes a single infiltration procedure. An indication of completeinfiltration is a distinct darkening in color of the underside of theleaf tissue. Following the vacuum infiltrations, the leaves and basketwere removed from the vacuum tank. The bags containing the vacuuminfiltrated leaves were allowed to gravity drain surface buffer for ˜10minutes, prior to centrifugation. The interstitial fluid (IF) wasrecovered from the vacuum

TABLE 7 GCB UF Experiment Fresh Total Protein Total Protein GCB TotalWeight Vol Conc. Protein GCB Yield Conc GCB Units/kg Sample/Fraction#(Grams) (ml) (mg/ml) (mg) (mg) (mg/g) (U/ml) (Units) Tissue IF 1,1025,874 0.223 1310 6.5 1.189 1,659 9,745,470 8,843,439 30K Concentrate1,102 875 0.593 519 6.39 0.471 10,947 9,578,575 8,691,992 Sp Activity %GCB Step Step Total Total nmol/hr % of total Recovery PurificationRecovery Purification Sample/Fraction# ((U)/mg) Protein = GCB (%) fold(%) fold IF 7,440 0.5 100 1 100 1 30K Concentrate 18,460 1.23 98.3 2.598.3 2.5 IF = interstitial fluidinfiltrated leaves by centrifugation (1,800×G, 30 minutes) using aHeine® basket centrifuge (bowl dimensions, 28.0 inches diameter×16.5inches).

Collected IF was filtered through a 50μ cartridge filter and then storedat 4° C., until the entire 100 kilograms of tissue was infiltrated. Thisprocess was repeated with the next set of four 5 kg bags (5×20 kg cyclestotal) until all the tissue was infiltrated. Additional buffer was addedduring each infiltration cycle to completely immerse the tissue.Alternatively, the leaf tissue can be re-infiltrated by placing theleaves back in the infiltration vessel in the same buffer used above andthe process repeated (IF-2). Additionally, the buffer may be drainedfrom the infiltration vessel (spent buffer) and may be pooled with the1st and 2nd IF fractions. Collectively, IF-1, IF-2 and spent bufferconstitutes the IF pool. The volume of interstitial fluid collected fromthe infiltrated leaf tissue was between 42–170% of the leaf tissue byweight depending on the number of infiltrations carried out.

Recombinant GCB was purified by loading the dilute interstitial fluid(feed stream) directly on a Pharmacia Streamline 200® column containingPhenyl Streamline® resin. Expanded bed chromatography enabled us tocapture, clarify and concentrate our protein in one step without theneed for centrifugation and/or microfiltration steps. The column wasequilibrated and washed until the UV-signal on the recorder returned tobaseline with 25 mM citrate, 20% ethylene glycol, pH 5.0; the boundenzyme was eluted with 25 mM citrate, 70% ethylene glycol. The elutedmaterial was sterile filtered by passing the eluted material through a 1sq. ft. 0.8 um Sartoclean GF® capsule followed by a 1 sq. ft. 0.2 umSartobran P® sterile filter (Sartorius, Corp.) and stored at 4° C. untilthe next chromatography step. The eluted material from 4–5 days ofPhenyl Streamline® chromatography runs was pooled together and furtherpurified on a cation exchange resin, SP Big Beads® (Pharmacia),equilibrated in 25 mM citrate, 75 mM NaCl, pH 5.0. GCB was eluted with astep gradient of 25 mM citrate, 0.4 M NaCl, 10% ethylene glycol, pH 5.0.All chromatography steps were carried out at room temperature. Theeluted material was sterile filtered by passing the eluted materialthrough a 1 sq. ft. 0.8 um Sartoclean GF® capsule followed by a 1 sq.ft. 0.2 um Sartobran P® sterile filter (Sartorius, Corp.) and stored at4° C.

Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 MPotassium Phosphate, 0.15% Triton-X100, 0.125% sodium taurocholate, 0.1%bovine serum albumin, pH 5.9 with and without CBE. Total glucosidaseactivity and rGCB activity were measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl glucopyranoside. Totalprotein was determined using the Bio-Rad Protein Assay® based on themethod of Bradford (Bradford, M. Anal. Biochem. 72:248; 1976).

Typically from 1 kilogram of field grown tobacco, expressing GCB, whereIF-1 alone was collected we obtained 435,000 units of GCB at a specificactivity of 2,745 units. The Unit/Kg increased to 755,000 with aspecific activity of 3,400 when IF Pool was collected (IF-1, IF-2 andspent buffer).

Table 8 below contains data that is representative of one week ofexperiments.

Example 10 Chopped Tissue Experiment

An experiment was carried out where 100 kilograms of MD609 leaf tissueof transgenic tobacco expressing the lysosomal enzyme glucocerebrosidasewas harvested off the stalks by hand, weighed and chopped into smallpieces to increase the surface area for buffer infiltration. Fivekilograms of leaves were placed into polyester bags (Filtra-Spec®,12-2-1053) and four×5 kg bags of leaves were placed into a metal basket.The metal basket containing the leaf material was placed in a 200 literMueller® vacuum tank containing ˜100 liters of buffered solution (0.1KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10 mM2-mercaptoethanol). A 70 lb. stainless steel plate was placed over theleaves/bags to assure complete immersion. A vacuum was pulled 695 mm Hg,held for 1 minute and then rapidly released. This vacuum infiltrationwas repeated for a total of two cycles. Following the vacuuminfiltrations, the leaves and basket were removed from the vacuum tank.The bags containing the vacuum infiltrated

TABLE 8 GCB Field Test Pilot Scale-P.SL Fresh Total Protein TotalProtein GCB Total Weight Vol Conc. Protein GCB Yield Conc GCB Units/kgSample/Fraction# (Grams) (ml) (mg/ml) (Mg) (mg) (mg/g) U/ml (Units)Tissue IF1,2&SB-Day 1 100,000.00 164,500 0.12 19740 49.24 0.197 44973,860,500 738,605 Phenyl Streamline eluded 100,000.00 37,600 0.04 15045.84 0.015 233 8,760,800 87,608 material IF1,2&SB-Day 2 100,000.00171,000 0.144 24624 51.41 0.246 451 77,121,000 77,121,000 PhenylStreamline eluted 100,000.00 42,500 0.036 1530 8.67 0.015 306 13,005,00013,005,000 material IF1,2-Day 3 100,000.00 95,500 0.547 52239 39.160.522 615 58,732,500 58,732,500 Phenyl Streamline eluted 100,000.0034,000 0.059 2006 22.05 0.02 973 33,082,000 33,082,000 material IF1-Day4 100,000.00 50,000 0.273 13650 20.23 0.137 607 30,350,000 30,350,000Phenyl Streamline eluted 100,000.00 35,800 0.046 1647 14.77 0.016 61922,160,200 22,160,200 material IF1-Day 5 100,000.00 86,000 0.348 2992835.03 0.299 611 52,546,000 52,546,000 Phenyl Streamline eluted100,000.00 40,700 0.065 2646 19.73 0.226 727 29,588,900 29,588,900material SP Big Beads-5 days of 500,000.00 191,650 0.053 10157 62.080.02 486 93,113,911 93,113,911 PSL runs SP Big Beads eluted 500,000.0017,000 0.043 731 48.35 0.001 4,266 72,529,928 72,529,928 material SpActivity % GCB Step Step Total Total nmol/hr % of total RecoveryPurification Recovery Purification Sample/Fraction# ((U)/mg) Protein =GCB (%) fold (%) fold IF1,2&SB-Day 1 3,742 0.25 100 1 100 1 PhenylStreamline eluded 5,825 0.39 11.9 1.6 11.9 1.6 material IF1,2&SB-Day 23,132 0.21 100 1 100 1 Phenyl Streamline eluted 8,500 0.57 16.9 2.7 16.92.7 material IF1,2-Day 3 1,124 0.07 100 1 100 1 Phenyl Streamline eluted16,492 1.1 56.3 14.7 56.3 14.7 material IF1-Day 4 2,223 0.15 100 1 100 1Phenyl Streamline eluted 13,457 0.9 73 6.1 73 6.1 material IF1-Day 51,756 0.12 100 1 100 1 Phenyl Streamline eluted 11,185 0.75 56.3 6.456.3 6.4 material SP Big Beads-5 days of 9,167 0.61 100 1 100 1 PSL runsSP Big Beads eluted 99,220 6.61 77.9 10.8 77.9 10.8 materialleaves were allowed to gravity drain surface buffer for ˜10 minutes,prior to centrifugation. The interstitial fluid (IF) was recovered fromthe vacuum infiltrated leaves by centrifugation (1,800×G, 30 minutes)using a Heine® basket centrifuge (bowl dimensions, 28.0 inchesdiameter×16.5 inches). Collected IF was filtered through a 50μ cartridgefilter and then stored at 4° C., until the entire 100 kilograms oftissue was infiltrated. This process was repeated with the next set offour 5 kg bags (5 cycles×20 kg cycles total) until all the tissue wasinfiltrated. Additional buffer was added during each infiltration cycleto completely immerse the tissue. In order to evaluate how much enzymewas recovered in the interstitial fluid, the tissue from which theinterstitial fluid was isolated was then homogenized in a Waring®blender with 4 volumes of the same infiltration buffer as above,centrifuged and the supernatant assayed for enzyme activity.

Recombinant GCB was purified by loading the dilute interstitial fluid(feed stream) directly on a Pharmacia Streamline 200® column containingPhenyl Streamline® resin. The column was equilibrated and washed untilUV-signal on recorder returned to baseline with 25 mM citrate, 20%ethylene glycol, pH 5.0 and then eluted with 25 mM citrate, 70% ethyleneglycol. All chromatography steps were carried out at room temperatureTable 9 below contains data from the chops experiment.

Example 11 Pilot Scale Purification of Alpha Galactosidase from theIntercellular Fluid of Nicotiana benthamiana

Actively growing Nicotiana benthamiana plants were inoculated withinfectious transcripts of a recombinant plant viral construct containingthe lysosomal enzyme alpha galactosidase gene wherein theα-galuctosidase gene contains a carboxy-terminal modification to thenucleotide sequence to enable secretion to the interstitial space.Systemically infected leaf tissue (1–2 kilograms) was harvested fromNicotiana benthamiana expressing alpha galactosidase 14 days postinoculation. The tissue was weighed and submerged with 2–4

TABLE 9 GCB Field Test Chops Fresh Total Protein Total Protein GCB TotalWeight Vol Conc. Protein GCB Yield Conc GCB Units/kg Sample/Fraction#(Grams) (ml) (mg/ml) (mg) (mg) (mg/g) (U/ml) (Units) Tissue IF1/Chops100,000.00 56,000 0.678 37946 10.42 0.379 279 15,624,000 156,240 PhenylStreamline 100,000.00 30,000 0.072 2147 9.38 0.021 469 14,070,000140,700 eluded material Tissue Homogenate 100,000.00 56,000 ND ND 15.080 404 22,621,081 226,211 Sp % GCB Activity % of Step Step Total Totalnmol/hr total Recovery Purification Recovery PurificationSample/Fraction# ((U)/mg) Protein = GCB (%) fold (%) fold IF1/Chops 4120.03 100 1 100 1 Phenyl Streamline 6,553 0.44 90.1 15.9 90.1 15.9 eludedmaterial Tissue Homogenate ND ND ND ND ND ND ND = not determinedvolumes of buffer (25 mM bis tris propane buffer, pH 6.0, 5 mM EDTA, 0.1M NaCl, 10 mM 2-mercaptoethanol) in an infiltration vessel that canaccommodate several kilograms of leaf tissue at one time. A perforatedmetal plate was placed on top of tissue to weigh down the tissue. Avacuum was pumped to 620–695 mm Hg for 30 seconds and then quicklyreleased. The tissue was rotated and the vacuum reapplied to achievecomplete infiltration which was confirmed by a distinct darkening incolor of the underside of the leaf tissue. Excess buffer on the tissuewas drained. The interstitial fluid was released from the tissue bycentrifuging the tissue in a basket rotor (10 in.×4.25 in. Depth,InterTest Equipment Services, San Jose, Calif./Biosource Design25-0611000) at 3800 RPM (2100×G) for 10–15 minutes. The interstitialfluid was collected by aspiration. In some instances only infected leaftissue was harvested. Alternatively, petioles and stems have beenharvested along with the leaf tissue for infiltration. The mid vein wasnot removed from the tissue prior to infiltration.

Alpha galactosidase was purified by loading the dilute intercellular(feed stream) directly onto a Pharmacia Streamline 25® column containingButyl Streamline® resin. Expanded bed chromatography enabled thecapture, clarification and concentration of the protein in one stepwithout the need for centrifugation and/or microfiltration steps. Thecolumn was equilibrated and washed until UV-signal on recorder returnedto baseline with 25 mM bis tris propane, pH 6.0 20% (NH₄)₂SO4 and theneluted with 25 mM bis tris propane, pH 6.0. The eluted material wasfurther purified on hydroxyapatite equilibrated with 1 mM NaPO₄ buffer,5% glycerol, pH 6.0 and eluted with either a 1–250 mM NaPO₄ buffer, 5%glycerol, pH 6.0 linear gradient or a step gradient. All chromatographysteps were carried out at room temperature.

Alpha galactosidase activity was measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl α-D galactopyranoside. Enzymeactivity was measured at 37° C. in a reaction mixture containing 5 mMmethylumbelliferyl α-D galactopyranoside, 0.1 M potassium phosphate,0.15% Triton-X100®, 0.125% sodium taurocholate, 0.1% bovine serumalbumin, pH 5.9. Total protein was determined using the Bio-Rad ProteinAssay® based on the method of Bradford (Bradford, M. Anal. Biochem.72:248; 1976).

From 1 kilogram of leaves, we typically obtain between 140–160 millionunits of alpha galactosidase at a specific activity of 800,000 unitsfollowing a single infiltration procedure (IF-1).

Table 10 below contains data that is representative of severalexperiments.

Example 12 Pilot Scale Purification of Glucocerebrosidase from the LeafInterstitial Fluid and of Recombinant Virus from the Leaf Homogenate ofField Grown Tobacco

Transgenic tobacco (MD609) expressing the lysosomal enzymeglucocerebrosidase was mechanically inoculated with a tobacco mosaicvirus derivative containing a coat protein loop fusion, TMV291, (Turpen,et.al., 1995, Bio/Technology 13:23–57). A total of 100 kg of transgenic,transfected leaf tissue was harvested from the field, five weeks postinoculation. The tissue was stripped off the stalks by hand and weighed.Five kilograms of leaves were placed into polyester bags (Filtra-Spec®,12-2-1053) and four×5 kg bags of leaves were placed into a metal basket.The metal basket containing the leaf material was placed in a 200 literMueller® vacuum tank containing ˜100 liters of buffered solution (0.1KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10 mM2-mercaptoethanol). A 70 lb. stainless steel plate was placed over theleaves/bags to assure complete immersion. A vacuum was pumped to 695 mmHg, held for 1 minute and then rapidly released. This vacuuminfiltration was repeated for a total of two cycles. Multipleapplications of the vacuum without isolating the interstitial fluidconstitutes a single infiltration procedure. An indication of completeinfiltration is a distinct darkening in color of the underside of theleaf tissue. Following the vacuum infiltrations, the leaves and basketwere removed from the vacuum tank. The bags containing the vacuuminfiltrated leaves were allowed to gravity drain surface buffer for ˜10minutes, prior to centrifugation. The interstitial fluid (IF) wasrecovered from the vacuum infiltrated leaves by centrifugation (1,800×G,30 minutes) using a Heine® basket centrifuge (bowl dimensions, 28.0inches diameter×16.5 inches). Collected IF was filtered through a 50μcartridge filter and then

TABLE 10 Pilot Scale alpha gal Fresh Total Protein Total Protein GalTotal Weight Vol Conc. Protein Gal Yield Conc Gal Units/kgSample/Fraction# (Grams) (ml) (mg/ml) (mg) (mg) (mg/g) (U/ml) (Units)tissue IF 2026 1,450 0.236 342 74.5 0.169 226,201 327,992,085161,891,454 Butyl Streamline 2026 300 0.392 118 74 0.058 1,085,873325,761,839 160,790,641 eluted material Hydroxyapatite eluted 2026 4700.076 36 54.2 0.018 507,640 238,590,619 117,764,373 material Sp Activity% Gal Step Step Total Total Nmol/hr % of total Recovery PurificationRecovery Purification Sample/Fraction# ((U)/mg) Protein = Gal (%) fold(%) fold IF 958,481 21.78 100 1 100 1 Butyl Streamline 2,770,084 62.9699.3 2.9 99.3 2.9 eluted material Hydroxyapatite eluted 6,679,469 151.8173.2 2.4 72.7 7 material IF = interstitial fluid extractionstored at 4° C., until the entire 100 kilograms of tissue wasinfiltrated. This process was repeated with the next set of four 5 kgbags (5 cycles×20 kg total) until all the tissue was infiltrated.Additional buffer was added during each infiltration cycle to completelyimmerse the tissue.

Recombinant GCB was purified by loading the dilute intercellular (feedstream) directly on a Pharmacia Streamline 200® column containing PhenylStreamline® resin. Expanded bed chromatography enabled the capture,clarification and concentration the protein in one step without the needfor centrifugation and/or microfiltration steps. The column wasequilibrated and washed until the UV-signal on the recorder returned tobaseline with 25 mM citrate, 20% ethylene glycol, pH 5.0; the boundenzyme was eluted with 25 mM citrate, 70% ethylene glycol.

The eluted material was sterile filtered by passing the eluted materialthrough a 1 sq. ft. 0.8 μm Sartoclean GF® capsule followed by a 1 sq.ft. 0.2 μm Sartobran P® sterile filter (Sartorius, Corp.) and stored at4° C. until the next chromatography step. The eluted material from 4–5days of Phenyl Streamline® chromatography runs was pooled together andfurther purified on a cation exchange resin, SP Big Beads® (Pharmacia),equilibrated in 25 mM citrate, 75 mM NaCl, pH 5.0. GCB was eluted with astep gradient of 25 mM citrate, 0.4 M NaCl, 10% ethylene glycol, pH 5.0.All chromatography steps were carried out at room temperature. Theeluted material was sterile filtered by passing the eluted materialthrough a 1 sq. ft. 0.8 um Sartoclean GF® capsule followed by a 1 sq.ft. 0.2 μm Sartobran P® sterile filter (Sartorius, Corp.) and stored at4° C.

Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 Mpotassium phosphate, 0.15% Triton-X100®, 0.125% sodium taurocholate,0.1% bovine serum albumin, pH 5.9 with and without CBE. Totalglucosidase activity and rGCB activity were measured by hydrolysis ofthe fluorescent substrate 4-methylumbelliferyl glucopyranoside. Totalprotein was determined using the Bio-Rad Protein Assay® based on themethod of Bradford (Bradford, M. Anal. Biochem. 72:248 (1976)).

The quantity remaining of virus present in IF extracted leaf tissue wasdetermined using homogenization and polyethylene glycol precipitationmethods. In addition, the amount of virus present in the pooled,interstitial fluid was determined by direct polyethylene glycolprecipitation. Final virus yields from precipitated samples wasdetermined spectrophotometrically by absorbance at 260 nm. (see Table11)

TABLE 11 Sample Virus Titer Pooled IF 0.004 mg virus/g fresh weight,0.010 mg virus/ml IF Homogenized leaf tissue 0.206 mg virus/g freshweight following IF Extraction

Table 12 contains the GCB recovery data from TMV transfected planttissue.

This example demonstrates the ability to extract two different productsfrom the same leaf tissue based upon extraction procedures thatspecifically target products localized in the apoplast and cytosol.

While the invention of this patent application is disclosed by referenceto the details of preferred embodiments of the invention, it is to beunderstood that this disclosure is intended in an illustrative, ratherthan limiting, sense. It is contemplated that modifications will readilyoccur to those skilled in the art, within the spirit of the inventionand the scope of the appended claims. It is further understood that theinstant invention applies to all proteins produced or capable of beingrecombinantly produced in plants, and is clearly not limited to thoseproteins specifically described herein.

Example 13 Large Scale Centrifugation of IF Fractions

The following example illustrates a scale-up procedure for theproduction of IF extract

TABLE 12 GCB Recovery From TMV Transfected Plants Fresh Total ProteinTotal Protein GCB Total Weight Vol Conc. Protein GCB Yield Conc GCBUnits/kg Sample/Fraction# (Grams) (ml) (mg/ml) (mg) (mg) (Mg/g) (U/ml)(Units) tissue IF1/Virus 100,000 40,000 0.383 15320 18.7 0.153 70128,055,851 280,559 Phenyl Streamline 100,000 25,000 0.024 600 6.66 0.006400  9,990,926  99,909 eluted material Sp % GCB Activity % of Step StepTotal Total nmol/hr total Recovery Purification Recovery PurificationSample/Fraction# ((U)/mg) Protein (%) fold (%) Fold IF1/Virus 1,831 0.12100 1 100 1 Phenyl Streamline 16,652 1.11 35.6 9.1 35.6 9.1 elutedmaterialusing a discontinuous batch method that will produce a constant streamof IF extract to downstream processing. This procedure consists of thefollowing elements:

1. Automated Whole Leaf Harvesting

2. Large Scale Continuous Infiltration

3. Large Scale Basket Centrifugation

There are at least two full-scale, whole leaf harvester designsavailable. One has been developed by R.J. Reynolds Company and has beenused at their Avoca facility in North Carolina. The other harvester hasbeen developed by University of Kentucky, Agricultural Engineeringdepartment and has been demonstrated for three seasons in Daviess CountyKentucky in commercial tobacco fields. These harvesters have shown thecapablilty to cut intact plants, strip-off whole leaves and separate theleaves and stem tissue at rates over several acres per hour. The leaveswill then be transported to the extraction facility in trailers.

The leaves will then be unloaded by mechanical conveyor and continuousweigh belt feeder into the vacuum infiltration system. Two systems havebeen designed. System 1 (FIG. 1) is a bulk tank. This tank isconstructed for full vacuum and is rotatable at low (less than 50) rpmso that all leaves are immersed in the infiltration medium. A vacuum iscreated by conventional mechanical vacuum pumps or by a steam ejector toa vacuum equal to 21 inches of water column pressure. The vacuum is thenreleased causing infiltration of the tissue. The vessel is then drainedto a secondary tank for buffer reuse and the leaf tissue is dischargedfrom the vessel via an auger in the bottom of the tank to a dischargeport and onto a conveyor. This conveyor transports the leaves to thebasket centrifuge via a weigh belt. The weigh belt insures that ameasured amount of material is added to the centrifuge for eachcentrifugations cycle. System 2 is a continuous vacuum infiltrationsystem. This system consists of large cylindrical tube that has aninternal auger conveyor (FIG. 2). The cylinder is placed at an angle.The cylinder is partially filled with the infiltration fluid. Thecylinder is under vacuum provided by conventional vacuum pumps or asteam ejector to approximately 21 inches of water column pressure. Leaftissue is added through a rotary valve that maintains the vacuum as itadds tissue. The leaf tissue is then immersed for a period of time inthe buffer as it travels up the tube, conveyed by the auger. Theinfiltrated leaves are discharged at the elevated end of the augerthrough another rotary valve. At this point the vacuum is released. Thistype of pressure vessel, equipped with rotary valves and an augertransport flight is adapted from a pressure vessel design by ChristianEngineering (San Francisco) that is used for continuous cooking of riceand other materials using steam pressure. Once discharged, the leavesare transported to the basket centrifuge via a conveyor equipped with aweigh belt. The weigh belt functions as stated above to insure theproper charge of material for each cycle of the basket centrifuge.

The basket centrifuge is a modification of a basic Sanborn (UPE) designfor the vegetable industry for dewatering salad greens after washing.The centrifuge is a basket design with a cone type spindle on the insideof the basket. The basket is a two piece design that accomplishes theseparation of the bottom plate from the cylinder via a hydraulic piston.The centrifuge is loaded at very low speed (i.e., low RPM or low Gforce) via a conveyor that is placed over the center of the basketequipped with the cone spindle. As the material drops from the conveyorit is deflected by the cone evenly upon the side of the perforatedbasket. When the charge of the leaves is complete the auger stops andthe basket is accelerated to 2000–2500×G for approximately 5–60 min. TheIF fluid is recovered from the centrifuge. At the end of thecentrifugation the basket is decelerated to a low rpm. The bottom of thebasket is separated from the sides (cylinder) by the action of thehydraulic piston. The leaf tissue is discharged to a conveyor, thebottom of the centrifuge is closed and the cycle is repeated. Thisdesign requires that a rotor and drive be designed that can be rated forthe higher G force. Typically the Sanbom type machines are only ratedfor 600 to 800 G. It is, however, within normal engineering parametersto construct such an upgraded machine for this unique application.

1. A method of extracting a desired protein from the interstitial fluidof a plant tissue, comprising: (a) harvesting leaf tissue; (b)submerging the leaf tissue in an infiltration buffer; (c) subjecting theleaf tissue and infiltration buffer to a substantial vacuum; (d)releasing the vacuum; (e) releasing the interstitial fluid; (f)capturing, clarifying and concentrating said desired protein in a singlestep by expanded bed chromatography wherein the protein is extracted. 2.A method according to claim 1 wherein steps c and d are repeated.
 3. Amethod according to claim 1 wherein the excess infiltration buffer isremoved.
 4. A method according to claim 1 wherein steps b through e arerepeated.
 5. A method according to claim 4 wherein the interstitialfluid is pooled.
 6. In a method of extracting a protein of interest froma plant tissue, comprising: (a) infiltrating a plant tissue with abuffer solution; (b) subjecting the plant tissue and buffer solution toa substantially vacuum environment; the improvement comprising, (c)applying expanded bed chromatography for, capturing, clarifying andconcentrating said desired protein.